Gram-positive bacterial pathogens pose a major threat to human health due to the development of resistance to most clinically relevant antibiotics. For this reason novel anti-infective targets need to be identified, developed and implemented in the fight against the rising tide of antibiotic resistance. A highly conserved protease essential for Gram-negative pathogens to survive in the host was found to be conserved in Gram-positive pathogens. An insertional knockout of the protease in S. pyogenes resulted in a temperature-sensitive phenotype and sensitivity to oxidative agents. The identification of this conserved family of proteins suggests that the protease is a true broad-spectrum anti-infective development target. The goals of this proposal are to clone the protease from S. aureus and construct an insertional knock-out in order to validate virulence in a mouse model of infection. The protease will be expressed and purified from E. coli and the preferred enzyme cleavage site identified through Phage Display and BioPanning. This data will allow the identification of natural protease targets in S. aureus and other Gram-positive pathogens. These targets will be utilized in the development of secondary screens and may also represent additional development targets. The target peptide sequence will be utilized in the design and development of a High Throughput Screening Assay based on target cleavage. Combinatorial libraries will be screened for small molecules inhibitors of enzymatic activity. Lead candidates will be validated based on several secondary in vitro and in vivo screens developed at SRL. Lead compounds will be optimized through combinatorial chemistry and prepared for pre-clinical toxicology testing. Lead compounds will be screened against E. faecalis, E. faeciurn, S. epidermidis and other important Gram-positive pathogens to validate the broad spectrum nature of the target.